We investigate the properties and regulation of the voltage-dependent calcium channels, and the coupling between calcium entry and transmitter release in isolated nerve endings from rat brain (synaptosomes). The long-term objective is to obtain a better understanding of the fundamental mechanisms which modulate calcium fluxes and transmitter release in presynaptic nerve terminals. In the present project: (1) The kinetic parameters, JCaMax and KCa, of the putative "fast" (phasic) and "slow" (tonic) Ca++ channels, and the kinetics of Ca++ entry inactivation in potassium depolarized synaptosomes will be determined as function of the membrane potential. The measurements will utilize a novel Quench-flow technique which permits the determination of the initital rates of Ca++ entry in the millisecond time range. (2) The intrasynaptosomal "depot" and "active" acetylcholine pools will be differentially labeled with (14C)ACh and (3H)ACh, respectively. The time-course of the evoked release of ACh from the respective pools will be studied by Quench-flow and rapid filtration techniques. The kinetics of ACh release will be compared with the kinetics of the biphasic Ca++ entry to determine more precisely the relationships between the biphasic Ca++ entry, biphasic transmitter release and the subcellular compartmentation of ACh in synaptosomes. (3) The mechanism of Ca++ entry inactivation, which delimits the phasic transmitter release, will be investigated. The following hypotheses will be tested: (a) the functional state of Ca++ channels in nerve terminals is determined by the cyclic-AMP dependent state of phosphorylation of the channel or its substructure, (b) dephosphorylation of the channel leads to its inactivation, and (c) dephosphorylation and channel inactivation may be mediated by an increase in intracellular ionized Ca++. Phospho-dephosphorylation of synaptosomal plasma membranes in intact synaptosomes and in purified synaptosomal plasma membrane fragments will be investigated within the time-constraints of the kinetics of Ca-entry inactivation. Quantitative autoradiography of polypeptide patterns on sodium-dodecyl- sulfate-polyacrylamide gell electrophoregrams will be employed to identify the endogenously 32P-phosphorylated polypeptide(s) whose phospho-dephosphorylation kinetics may correlate with the functional states of Ca++ channels in synaptosomes.